Development and testing of novel recombinant pnemococcal glyconjugate vaccines

Streptococcus pneumoniae, or the pneumococcus, can cause life-threatening diseases such as pneumonia, septicaemia, meningitis and frequently causes ear infections in children which can lead to hearing loss. S. pneumoniae is responsible for significant morbidity and mortality worldwide and by conservative estimates pneumococcal infections cause over one million deaths of children annually. An inexpensive, broad-range, long-lasting pneumococcal vaccine is desperately required.

A defining characteristic of a successful vaccine is the ability to evoke long-lasting protective immunity with minimal side effects. The most successful human vaccines are often glycoconjugate as the combination of a protein coupled to a sugar glycan induces both a T-cell dependent and independent immune response evoking a protective and lasting immunity. Examples of currently licensed human glycoconjugate vaccines include those against Haemophilus influenzae, Neisserria meningitidis and some Streptococcus pneumoniae strains, in which glycans are chemically coupled to immunogenic carrier proteins.

Traditional glycoconjugate vaccine design by chemical conjugation requires that the glycan from the pathogenic organism be isolated, detoxified by stripping out surface components, and still be present in sufficient amounts to be chemically coupled to a protein. The procedures involve harsh chemical treatments, are time consuming and expensive. In addition, the material generated at each step needs to be verified for purity, and variation between batches of glycoconjugate vaccine is common. Current licensed pneumococcus glycoconjugate vaccines are problematic as they only cover a fraction of all S. pneumoniae strains. Although there are vaccine candidates based on conserved proteins, these vaccines often do not produce long-term protection that is especially required to immunize the main target population, children. Ideally, a glycoconjugate vaccine based on conserved pneumococcal proteins coupled to the capsular polysaccharide glycan should be produced, but to date this has proved technically challenging.

Recently, we (and collaborators) have developed a new approach for constructing glycoconjugate vaccines involving cloning all components in the widely used "work-horse" microbe E. coli. The recombinant process is termed Protein Glycan Coupling Technology (PGCT) and involves processing the candidate protein and glycan in plasmid vectors in E. coli along with a coupling enzyme to produce an inexhaustible supply of vaccine. PGCT can produce purified vaccine in a one-step purification procedure, which reduces costs, and because multiple combinations of protein and glycans can be coupled together, a greater flexibility in the range of vaccines can be generated and tested. We will use PGCT to produce and test six outstanding protein candidates coupled to different combinations of pneumococcal capsular polysaccharide. These vaccines will be tested in the murine pneumococcal infection model for their relative protection against an otherwise lethal dose. Additionally, the vaccines will be tested for their effect on the carriage of pneumococci in the murine model. The new vaccines generated in this study will also be compared to the efficacy of existing pneumococcal vaccines such as Prevnar13. Data between experiments will be evaluated to derive the most efficacious glycoconjugate vaccine combination produced by PGCT. Additionally, the development of PGCT in this study will provide the expertise and knowledge base to make the technology more widely applicable to construct further S. pneumoniae glycoconjugate vaccines and vaccines against other important infectious agents.

Streptococcus pneumoniae causes pneumonia, septicaemia and meningitis, and globally one million children die of pneumococcal disease each year. Current pneumococcal glycoconjugate vaccines require both purification of the capsule glycan from the pathogen and its chemical coupling to a suitable protein carrier. These are costly and only protect against some S. pneumoniae serotypes, which can promote capsule switching and vaccine escape strains.

The Wren group has pioneered an approach termed bacterial Protein Glycan Coupling Technology (PGCT) whereby combinations of recombinant protein/glycan structures are produced in E. coli exploiting a toolbox of bacterial oligosaccharyltransferases. This recombinant approach promises maximum flexibility in terms of glycoconjugate design that should produce an unlimited and purified supply of vaccine at low cost.

The Mitchell group has demonstrated that inactivated pneumolysin protein can provide a mucosal immune response against a broad range of pneumococci, but requires a boost to produce a lasting response. Additionally other conserved protein candidates have been shown to be effective in the murine infection model.

We hypothesise that addition of a capsule determinant(s) to a protein carrier would produce a "best of both worlds" pneumococcal vaccine in terms of efficacy and protection range. The capsule(s) will be enzymatically coupled to six conserved pneumococcal protein or fusion protein vaccine candidates.

In this study, we will combine the expertise to engineer recombinant glycoconjugate vaccines using PGCT and test for protection in the pneumococcal murine infection model. The study will produce a novel serotype-independent pneumococcal glycoconjugate vaccine that will induce cellular and humoral immunity. The successful production of glycoconjugate vaccines using PGCT should have wide applications to construct further S. pneumoniae glycoconjugate vaccines and vaccines diverse pathogens.

Given that vaccination is a core public health measure in reducing the infectious disease burden, the proposed studies could make a major impact for the health and well-being of both animals and humans. This is particularly the case for pneumococcal disease where a reported estimate of over 1 million children die each year and at least an equivalent are burdened with the disease through long-term complications. A cheap efficacious vaccine is desperately required. The approaches described in the proposal using bacterial Protein Glycan Coupling Technology (PGCT) promises to break new ground in pneumococcal vaccination to produce an effective and inexpensive glycoconjugate vaccine. This multidisciplinary research proposal fits squarely into several priority research areas identified by the MRC, in terms of reducing the burden of childhood diseases, vaccine candidates and the design of novel treatments.

A longer-term impact would be to develop the PCGT for the modification of human proteins used as therapeutics in the Pharmaceutical industry. Additionally the characterization of glycan biosynthetic pathways (such as the S. pneumoniae capsule loci) will impact synthetic biology approaches providing cassettes of genes that encode known glycan structures that could have diverse functions.

We propose to disseminate our studies through publication in international peer-reviewed journals (where the applicants have a strong record) and by poster and oral presentations at major national and international meetings. The applicants have a track record of communicating the results of their research to the public and the mass media. Where possible these links will be used to promote this research.

The potential impact of the research will also be realised though our respective technology transfer offices, material transfer agreements and patents. We will continue to collaborate with Glycovaxyn, a 40+SME dedicated to the design and manufacture of glycoconjugate vaccines using PGCT. Glycovaxyn and other potential biotechnology companies with an interest in glycoengineering and/or vaccine development would provide the route to realize the potential of the proposed work subject to appropriate licensing agreements. The intellectual property for the PLY componentl vaccine has been licensed to Pfizer.

Currently, we have an international lead in the development of pneumococcal vaccines and recombinant glycoconjugate vaccines and require completion of the proposed study to maintain this lead. The research program should give the UK a significant boost in this important and topical health care issue and will contribute to the UK science base.